Abstract The ?brain in a dish? is an ideal in vitro system to model human neuronal behaviors in health and disease. In this study, we aim to establish benchmarks of Alzheimer's disease (AD) neuropathology formation in three dimensional (3D) neuronal cultures derived from AD subjects. Evolving cellular models for AD include a 3D human neuronal culture system which depends on overexpression of two AD genes to replicate AD neuropathology. We have recently advanced this model by creating a 3D neuronal culture using induced pluripotent stem cells (iPSC) derived from AD patients. This novel model is the first to use AD iPSC-derived 3D neurons that do not overexpress exogenous genes. When fully characterized, this model will substantially advance AD research and drug development. In this study, we propose to characterize the generation of tau- containing neurofibrillary tangles (NFT) and amyloid ? protein (A?)-containing neuritic plaques, the two pathological hallmarks of AD, and test the dependence of this pathology on a select group of upstream facilitators using standard techniques for selective knockdown. In preliminary studies, we have found increased levels of tau and neurofilament light polypeptide (NfL) in AD neurons, consistent with published analyses of blood and cerebrospinal fluid (CSF) of AD patients. We have also characterized post-mortem brain tissue and identified a tau binding protein S100B that was significantly increased in brain tissue from AD patients. Our aims reflect our hypotheses that 1) AD patients' iPSC-derived 3D neuro-spheroids recapitulate the molecular neuropathology of AD brains, and 2) the molecular neuropathology is responsive to manipulation of established and experimental upstream factors. First, we will aim to detect A? and tau pathology in iPSC- derived 3D human neurons and compare their properties to those in postmortem brain samples. We will characterize biochemical properties of A? aggregates and quantify A? peptides, and we will also characterize/compare ptau profiles. We will determine whether 3D neuro-spheroids from AD subjects have more A? and ptau than those from control subjects. We will also calculate ratios of A?42/A?40 and ptau/tau and determine whether higher ratios are found in 3D neuro-spheroids from AD patients. We will determine whether A?/tau pathologies are present in iPSC-derived 3D neuro-spheroids. Second, we will aim to validate proteins facilitating pathogenesis in 3D neuro-spheroids by chemical inhibition and genetic knock-down. We will determine if blocking BACE1 and S100B reduces A?/tau pathology formation in iPSC-derived 3D neuro- spheroids. We will also determine if reduction of NfL in 3D neuro-spheroids deters A?/tau pathology formation. These studies will validate this in vitro model and establish standards for future utilization. Future studies will target these facilitators for therapeutic intervention.